This research proposal concerns the transport of amino acids into prokaryotic cells. We have used genetic and biochemical approaches to identify four components, livJ, livK, livH, and livG, of high affinity leucine transport in E. coli. We are now applying cloning and DNA-sequencing techniques to these studies. A plasmid containing the entire leucine transport regulon has been prepared for in vitro synthesis and in vivo expression of the leucine transport components. Additional plasmids are being prepared which code for each of the two periplasmic leucine-binding proteins, leucine-specific and LIV-binding proteins. These periplasmic proteins are secretory proteins, and we are using an in vitro synthesis system to study the processing of the binding proteins to the mature forms. The processing enzyme will be isolated and purified. The DNA sequence of the N-terminal portion of the leucine-binding protein genes will give us the amino acid sequence of the signal peptides of the precursor forms. The techniques, combined with two-dimensional gel electrophoresis, will be used to identify and characterize the membrane-bound components, coded for by livH and livG. We are also applying in vitro transcription and coupled transcription/translation to elucidate the genetic regulatory mechanisms of livJ and livK expression.